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Magnetic susceptibility and proton magnetic resonance spectra are reported for the oxidized and reduced forms of the iron-sulfur protein Bacillus polymyxa ferredoxin I. The magnetic susceptibility of the oxidized form indicates antiferromagnetic exchange coupling between component iron atoms that is quantitatively similar to that observed for the clostridial ferredoxins and for the [(C(2)H(5))(4)N](2) [Fe(4)S(4)(SCH(2)C(6)H(5))(4)] analog. Contact-shifted resonances observed in the proton-magnetic-resonance spectra of oxidized and reduced forms of the B. polymyxa protein can be correlated with the contact-shifted resonances of corresponding redox forms of the clostridial ferredoxins. Characteristics of the contact-shifted resonances observed in partially reduced B. polymyxa ferredoxin I are compatible with a "slow" rate of electron exchange between redox forms, which suggests that the "fast" electron exchange earlier observed in the eight-iron clostridial ferredoxins may derive from an intramolecular component.

Magnetic susceptibilities of both reduced and oxidized ferredoxin from Clostridium pasteurianum were obtained in solution. Whereas the reduced form exhibits a Curie law behavior, the magnetic susceptibility of oxidized ferredoxin in fact increases with temperature and suggests extensive antiferromagnetic exchange coupling between the component iron atoms. Contact-shifted resonances are observed for both forms of ferredoxin that are attributed to the beta-CH(2) protons of the eight cysteine residues. A model based on these results is presented.

The rapid development of NMR spectroscopy has been characterized by a succession of discrete, significant advances in instrumentation, as well as by less dramatic but cumulatively important improvements in instrument performance, experimental techniques, spectral analysis, and theory. Most significant are the advances in magnet technology, which within 13 years increased the available field strengths from 7.04 to 51.7 kilogauss (with corresponding increase in the PMR frequency from 30 to 220 megahertz). Great improvements in spectrometer stability and in the coupling of spectrometers with on-line computers have so improved sensitivity that some nuclei possessing less favorable NMR characteristics can now be studied.

The synthetic analog approach has been applied to a clarification of the active sites of 2Fe-2S(*) proteins. The compound (Et(4)N)(2)[FeS(SCH(2))(2)C(6)H(4)](2), derived from o-xylyl-alpha,alpha'-dithiol, has been prepared and its structure has been determined by x-ray diffraction. The centrosymmetric anion contains two tetrahedrally coordinated ferric ions bridged by two sulfide ions and separated by 2.70 A. Comparison of electronic, Mössbauer, and proton magnetic resonance spectra and magnetic susceptibility of the anion with the corresponding properties of the oxidized forms of the proteins reveals significant degrees of similarity. The anion also exhibits the essential redox capacity of the proteins. We conclude that [FeS(SCH(2))(2)C(6)H(4)](2) (2-) possesses the same total oxidation level and electronic configuration as the active sites of the oxidized proteins, and that its structure provides a feasible representation of the minimal structure of the active site. [FeS(SCH(2))(2)C(6)H(4)](2) (2-) is thus the first well-defined synthetic analog of the active sites of two-iron ferredoxins.

The compound (Et(4)N)(2)[Fe(4)S(4)(SCH(2)Ph)(4)] has been prepared and its structure determined by x-ray diffraction. The Fe(4)S(4) core of the anion possesses a configuration of D(2d) symmetry that is closely related to the Fe(4)S(4) active-site structures of the high-potential iron protein from Chromatium and the ferredoxin from Micrococcus aerogenes. Electronic properties of the tetrameric anion have been partially characterized by measurement of proton magnetic resonance, Mössbauer, photoelectron, and electronic spectra, and magnetic susceptibility. Comparison of corresponding properties of [Fe(4)S(4)(SCH(2)Ph)(4)](2-) and the proteins implies that the oxidation levels of the synthetic tetramer, the reduced form of the high-potential protein, and the oxidized form of the 8-Fe ferredoxins are equivalent. The tetramer possesses the one-electron redox capacity associated with the 4-Fe centers of the ferredoxins. The structural and collective electronic features of [Fe(4)S(4)(SCH(2)Ph)(4)](2-) reveal it to be the first well-defined synthetic analogue of the active site of an iron-sulfur protein.

Contact-shifted resonances have been detected in the pmr spectra of both oxidized and reduced forms of spinach and parsley ferredoxins. These resonances are assigned to the beta-CH(2) protons of four cysteine residues that are thought to bind the iron-sulfur redox center to the polypeptide chain. Temperature dependences of contact shifts reveal that the two iron atoms are antiferromagnetically coupled in both redox forms of each of these proteins. Thermal population of magnetic states gives rise to the contact shifts observed in the formally diamagnetic oxidized forms of these ferredoxins and accounts for the failure of contact shifts in the reduced forms exhibit to a Curie Law temperature dependence. It appears that the unpaired electron of reduced spinach and parsley ferredoxin is unequally distributed over the two iron centers. Valence states for the iron pairs of high-spin Fe(+3)-Fe(+3) and Fe(+2)-Fe(+3) for the oxidized and reduced forms, respectively, are compatible with the nmr results.

Contact-shifted nuclear magnetic resonances, arising from molecular paramagnetism, have been observed in both reduced and oxidized forms of the high-potential iron protein (HiPIP) isolated from Chromatium. Contact shifts of the reduced, formally diamagnetic form increase with temperature, indicating antiferromagnetic exchange coupling of the component iron atoms with thermal population of a magnetic state. In the oxidized form of HiPIP (formally S = 1/2), contact-shifted resonances attributed to the beta-CH(2) groups of two cysteine residues display approximate Curie law behavior, while contact-shifted resonances assigned to the two other cysteine residues continue to exhibit a temperature dependence characteristic of antiferromagnetic exchange coupling. A cluster model for the redox center of Chromatium HiPIP that appears compatible with the PMR and preliminary x-ray results(4, 11) is discussed.

Single-stranded nucleic acids in aqueous media exhibit proton magnetic resonance spectra with acceptable signal-noise ratios if multiple scans of the spectra are stored in and subsequently read out of a computer of average transients. Partial ordering of the adenine bases of polyadenylic acid in solution is reflected in contributions to the chemical shifts that are removed on "melting." More significantly, secondary structure such as occurs in complexes ofpolyA and polyU and in double-stranded DNA is manifested in proton magnetic resonance spectrabelow the "melting" temperature by severe resonance line broadening which results from anisotropic nuclear dipole-dipole interactions. The proton resonances are narrowed in and above the "melting" region, and measurement of integrated resonance intensities can give insight into quantitative aspects of hybridization, "melting," and other nucleic acid interactions.

We studied the conformation of a series of gamma-ethyl-L-glutamate oligopeptides by circular dichroism and 220 MHz nuclear magnetic resonance spectroscopy. By use of the first technique we noted enhancement of the n --> pi(*) and splitting of the pi --> pi(*) transitions commencing with the heptamer in trimethylphosphate and trifluoroethanol. With the second method we found changes in chemical shifts for the amide protons consistent with the onset of helicity at the heptamer in the solvents noted above. When DMSO-d(6) is used as a solvent, no such chemical shift changes occur because the oligopeptides do not assume helical conformations.